Compositions, methods and related uses for cleaving modified DNA

ABSTRACT

Compositions, methods and related uses are provided relating to cleaving modified DNA. For example, a set of DNA fragments obtainable by enzymatic cleavage of a large DNA is described where at least 50% are similarly sized and have a centrally positioned modified nucleotide. In addition, an enzyme preparation is provided that includes one or more enzymes that recognize a modified nucleotide in a DNA and cleave the DNA at a site that is at a non-random distance from the modified nucleotide. The one or more enzymes are further characterized by an N-terminal conserved domain with greater than 90% amino acid sequence homology to WXD(X) 10 YXGD. The related uses include creating a methylome, methods of purifying DNA fragments containing a modified nucleotide and diagnostic applications.

CROSS REFERENCE

This application is a divisional application of U.S. patent applicationSer. No. 12/644,666, filed on Dec. 22, 2009, which application claimspriority from U.S. Provisional Application Ser. No. 61/140,586 filedDec. 23, 2008 and Ser. No. 61/267,617 filed Dec. 8, 2009, hereinincorporated by reference.

GOVERNMENT RIGHTS

This invention was made with Government support under contract5R44GM095209-03 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

BACKGROUND

DNA cleaving enzymes associated with methyltransferases are widelypresent in the prokaryotic genomes. The DNA cleaving enzymes typicallyconsist of restriction endonucleases, which protect host cells frominvading DNA (e.g., bacteriophages) by cleaving DNA at defined sites,and DNA methyltransferases, which protect host DNA from being degradedby methylating a specific base within the restriction endonuclease sites(Roberts, et al. Nucleic Acids Res 35: D269-270 (2007)). Hence, theserestriction endonucleases are termed methylation-sensitive.

While modified bases in prokaryotes and phage DNA play a role inprotecting the genome against cleavage by restriction endonucleases,methylated cytosine (m5C) is involved in gene expression of themammalian genome. Techniques for identifying methylated DNA arecumbersome and experimentally difficult to implement in a reproduciblefashion. Two approaches are commonly used. One involves the use ofrestriction enzymes like HpaII and MspI which are differently sensitiveto cytosine methylation. For example, HpaII endonuclease is blocked bymethylation of either of the two cytosines within the CCGG recognitionsite, but its isoschozimer, MspI, is blocked only when the outer C ismethylated. It will cleave DNA when the inner cytosine is modified. Thesecond method involves bisulfite modification of the unmethylatedcytosine residues followed by selective amplification and sequencing ofthe remaining DNA. In this method, methylated cytosines are resistant tothe treatment. This method is not easy to optimize and involves acomplicated chemical modification step followed by amplification using acomplicated set of primers. The method is widely used in the absence ofsimpler alternative approaches.

SUMMARY

In an embodiment of the invention, a set of double-strandedoligonucleotide fragments are provided that are obtainable by enzymaticcleavage of a large DNA wherein the large DNA contains one or moremodified nucleotides and may be derived from mammalian cells, morespecifically human cells. At least 50% of the fragments in the setshould preferably be of a similar size and preferably contain acentrally positioned modified nucleotide. One or more fragments may beisolated from the set. The large DNA may be at least 100 nucleotides inlength; the modified nucleotide is, for example, a modified cytosinesuch as a methylated cytosine or a hydroxymethylated cytosine and amodified cytosine may be proximate to a guanine to form a CpG or a CNG.However, a modified cytosine may be alternatively located next toanother cytosine, an adenine or a thymidine. Oligonucleotide fragmentsin the set may preferably be less than 60 nucleotides long, for example28-36 nucleotides; and/or the modified nucleotide, in particular,cytosine may be located within 30 nucleotides from either end of thefragment.

In an embodiment of the invention, an enzyme preparation is providedthat is characterized by one or more enzymes that recognize a modifiednucleotide in a DNA such that each enzyme is capable of cleaving the DNAat a site that is a non-random distance from the modified nucleotide.More particularly, the non-random distance between the cleavage site andthe modified nucleotide may be characteristic for the enzyme so as togenerate a set of fragments of the type described above. The one or moreenzymes are further characterized by an N-terminal conserved domain withgreater than 90% amino acid sequence homology with WXD(X)₁₀YXGD, moreparticularly with greater than 90% amino acid sequence homology withWXD(X)₆G(X)₃YXGD(X)₁₀₋₁₅GN(X)₂L X₁₀₋₂₀PX₃F.

In an embodiment of the invention, the one or more enzymes in the enzymepreparation are further defined by a recognition domain and a cleavagedomain within a single open reading frame. The cleavage domain may havean amino acid sequence which has greater than 90% amino acid sequencehomology to FEX₂₀₋₃₀DX₂₋₄DX₁₉₋₂₂(Q/E)XK. In addition, at least one ofthe enzymes may have an amino acid sequence homology of greater than 90%to any of the sequences identified as SEQ ID NOS:7-22. Additionally, oneor more of the enzymes may be covalently or non-covalently linked orfused to a protein affinity tag or other tag. Examples of suitableaffinity tags include a chitin-binding domain, maltose-binding domain,an antibody and a His tag. In addition, the one or more enzymes may berecognized by an antibody with binding specificity for an amino acidsequence comprising WXD(X)₁₀YXGD. Additionally, the preparation mayinclude an activator DNA.

In an embodiment of the invention, an enzyme preparation is providedthat includes one or more enzymes that recognize a modified nucleotidein a DNA such that each enzyme is capable of cleaving thedouble-stranded DNA at a site that is at a non-random distance from themodified nucleotide, more particularly where the distance between thecleavage site and the modified nucleotide is characteristic for theenzyme, thereby generating a set of fragments. The set of fragments maybe of similar size if the DNA contains a modified nucleotide on eachstrand of the duplex at approximately opposing positions or may be ofvarying size for hemi-modified DNA. The one or more enzymes may befurther characterized by an N-terminal conserved domain with greaterthan 90% amino acid sequence homology to WXD(X)₁₀YXGD.

In an embodiment of the invention, an antibody is provided that iscapable of recognizing and binding to an N-terminal domain of an enzymedescribed above.

In an embodiment of the invention, a method is provided which comprisescleaving a large DNA containing one or more modified nucleotides with acomposition described above and obtaining a set of oligonucleotidefragments. The method may further include separating the set ofoligonucleotide fragments from uncleaved DNA and additionally mayinclude sequencing from the separated set of fragments at least onefragment to determine the location of one or more modified nucleotidescontained within at least one fragment. The method may include analyzingsome or all of the oligonucleotide fragments for the presence andlocation of one or more modified nucleotides in the large DNA bysequencing or other means and mapping the sequences onto a genome ormethylome map to determine the location of modified nucleotides.

In an embodiment of the invention, a method is provided for identifyingan enzyme such as described above that includes searching a sequencedatabase using a sequence selected from the group consisting of SEQ IDNO:7-22 and variants thereof, and identifying additional sequenceshaving an N-terminal region characterized by a consensus sequence ofWXD(X)₁₀YXGD. The method may include the further step of identifying aC-terminal end comprising a catalytic domain with a consensus sequenceof FEX₂₀₋₃₀DX₂₋₄DX₁₉₋₂₂(Q/E)XK, more particularly FE(X)₂A(X)₁₅₋₁₈T/SX₄DGGXDX₂G/LX₁₅₋₂₀E/QAK.

In an embodiment of the invention, a method is provided for isolatingfrom a mixture of DNA fragments those DNA fragments containing one ormore modified nucleotides, the mixture resulting from enzyme cleavage ofa large DNA containing at least one modified nucleotide. The method mayinclude adding to the mixture an immobilized or labeled affinity-bindingmolecule that is capable of binding selectively those fragmentscontaining a modified nucleotide. Alternatively, those fragmentscontaining a modified nucleotide may be size-separated from thosefragments that do not contain a modified nucleotide. An example of anaffinity-binding molecule is an enzyme preparation described above,wherein the one or more enzymes in the enzyme preparation have beenmutated so as to lack enzyme cleavage activity and wherein the mutatedenzyme is immobilized on a solid surface so as to bind the DNA fragmentscontaining one or more modified nucleotides. Other examples ofaffinity-binding molecules include antibodies, inactivated T4glucosyltransferase and the methyl-binding domain of a cell protein suchas DNMT1. These molecules may in turn be fused to any of achitin-binding domain, a maltose-binding domain or a biotin molecule forexample and hence, bind to a suitable column.

In another embodiment of the invention, a method is provided foridentifying a present or future phenotypic property in a cellpreparation or tissue sample from a pattern of modified nucleotides. Themethod includes cleaving into fragments a large DNA from a cellpreparation or tissue by means of an enzyme preparation described above;and comparing a location for modified nucleotides in the fragments witha pattern of modified nucleotides in a control DNA so as to determine apresent or future phenotypic property.

In another embodiment of the invention, the above method furthercomprises contacting the cleavage fragments with an affinity-bindingmolecule capable of binding the modified nucleotide or by means ofelectrophoresis or other means known in the art capable of effectingsize separation. The binding moiety may include an enzyme preparation asdescribed above, wherein the enzyme cleavage activity has beeninactivated by conventional means. Thus, fragments with a modifiednucleotide may be separated from fragments lacking a modifiednucleotide. The above method may additionally include identifying on amethylome or a genome a location for the one or more modifiednucleotides in the immobilized cleavage fragments. The location may bedetermined by sequencing the separated fragments.

In another embodiment of the invention, a method is provided fordetermining the location of at least one modified nucleotide in a largeDNA. The method includes: cleaving a large DNA with an enzymepreparation described above; obtaining a set of oligonucleotide cleavageproducts containing at least one modified nucleotide; and determiningthe location of the at least one modified nucleotide in a sequence ofthe large DNA by for example sequencing the set of oligonucleotidecleavage products. The number of oligonucleotide fragments forsequencing in the set may depend on whether the set is derived fromcloned DNA or from repeats in which it may be sufficient to sequence asubset of fragments or on whether the set is expected to contain uniquesequences in which it may be desirable to sequence substantially all thefragments in the set.

In an embodiment of the invention, a method is provided for obtaining apurified preparation of fragments containing one or more modifiednucleotides that includes contacting a mixture of DNA fragments in whichone or more the fragments contain at least one modified nucleotide withan immobilized affinity-binding protein capable of binding covalently ornon-covalently to the DNA fragment. An example of an affinity-bindingprotein is a mutated enzyme in the enzyme preparation described above,wherein the enzyme cleavage activity has been inactivated. The methodmay further include binding the one or more fragments containing atleast one modified nucleotide to the binding protein; and obtaining apurified preparation of fragments containing one or more modifiednucleotides.

In an embodiment of the invention, a kit for generating oligonucleotidefragments containing a modified nucleotide is provided that includes anenzyme preparation described above in a container with instructions foruse. The kit may further include an activator molecule.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D show the MspJI RM system.

FIG. 1A shows the genomic segment of Mycobacterium sp. JLS encoding theMspJI RM system. NCBI annotations for open reading frames are: Mjls0821,putative helicase; Mjls0822 (MspJI), restriction endonuclease; Mjls0823(V.MspJIP), DNA mismatch endonuclease vsr; Mjls0824 (M.MspJI), DNAcytosine methyltransferase.

FIG. 1B shows the schematic domain structure of the MspJI enzyme family.The N-terminal domain is defined here as about 50% of the proteinsequence upstream of the remaining C-terminal domain.

FIG. 1C shows the conserved motif in the N-terminal domain.

FIG. 1D shows the conserved motif in the C-terminal domain.

FIGS. 2A-2D show MspJI activity in different in vitro reactions.

FIG. 2A shows modification-dependent enzyme activity for MspJI.

-   -   Lane 1, 1 μg of pBR322(dcm+) DNA only;    -   Lane 2, 1 μg of pBR322(dcm+)+0.8 μg MspJI;    -   Lane 3, 1 μg of pBR322(dcm+)+0.8 μg MspJI+10 units of BstNI;    -   Lane 4, 1 μg of pBR322(dcm+)+10 units of BstNI only;    -   Lane 5, 1 μg of pBR322(dcm−) DNA only; and    -   Lane 6, 1 μg of pBR322(dcm−)+0.8 μg MspJI.

All reactions were incubated at 37° C. for 1 hour and resolved on a 1%agarose gel. Lanes 3 and 4 show that MspJI does not cut any sites notcut by BstNI. Thus, on this substrate which is methylated at CmC A/T GG,only modified Dcm sites are cut by MspJI. All Dcm sites are cut by BstNIwhich is not sensitive to Dcm methylation.

FIG. 2B shows MspJI digestion on pBR322(dcm−) methylated at other sites.All reactions were done in 50 μl volume at 37° C. for 2 hours andresolved on 1% agarose gel. 0.8 μg of MspJI were used in all reactions.

-   -   Lane 1, 1 μg of M.MspI (C^(m)CGG) modified pBR322;    -   Lane 2, 1 μg of M.HaeIII (GG^(m)CC) modified pBR322;    -   Lane 3, 1 μg of M.HpaII (C^(m)CGG) modified pBR322;    -   Lane 4, 1 μg of M.HhaI (G^(m)CGC) modified pBR322; and    -   Lane 5, 1 μg of M.AluI (AG^(m)CT) modified pBR322.

FIG. 2C shows MspJI digestion on hydroxymethylcytosine-containing DNA.All reactions were carried out in 50 μl volume at 37° C. for 1 hour.

-   -   Lane 1, 1 μg of T4 wild-type (wt) DNA with glucosylated        hydroxymethylcytosine;    -   Lane 2, 1 μg of T4 gtu DNA with hydroxymethylcytosine;    -   Lane 3, 1 μg of T4 wt DNA+10 units of McrBC;    -   Lane 4, 1 μg of T4 gt DNA+10 units of McrBC;    -   Lane 5, 1 μg of T4 wt DNA+20 units of MspI;    -   Lane 6, 1 μg of T4 gt DNA+20 units of MspI;    -   Lane 7, 1 μg of T4 wt DNA+0.8 μg of MspJI; and    -   Lanes 8 through 11, 1 μg of T4 gt DNA with 2-fold        serially-diluted MspJI starting at 1.6 μg (Lane 8).

FIG. 2D shows the effect of different amounts of a DNA activator onMspJI activity. From Lanes 1 to 5, each reaction contains 1 μg (0.35pmol) pBR322 and 1.6 pmol MspJI. Lanes 1-4 show a titration (40, 20, 10,5 pmol) of the DNA activator containing methylated CCWGG sites. Lane 5shows pBR322-digestion using MspJI without DNA activator. Lane 6 showspBR322-digestion using BstNI (CCWGG).

FIG. 3 shows a schematic diagram of MspJI's cleavage activity onfully-methylated DNA. The double-stranded cleavage can happen at eitherside of the methylated site. The cleavage is on the 3′ side of therecognized methylated base. In this figure, when the top strandmethylated cytosine is recognized, MspJI cleaves on the right side; whenthe bottom strand methylated cytosine is recognized, MspJI cleaves onthe left side. The distances from the cleavage sites to the recognizedmethylated cytosine are fixed. For example, when top strand methylatedcytosine is recognized, the bottom strand nicking site is 16 nucleotidesaway from it and the top strand nicking site is 12 nucleotides away fromit.

FIGS. 4A-4D show MspJI cleavage on fully-methylated and hemi-methylatedoligo substrates, fractionated on a denaturing gel.

FIG. 4A shows the expected enzyme cleavage sites (designated Rt, Rb, Lt,Lb) in a synthetic double-stranded oligonucleotideTGGTAATAATAAGGTTGAGGACTTTTTCCGGATGCCCGGAATGGGTTCAAAGG (SEQ ID NO:1). The3′ end of the top strand or the 5′ end of the bottom strand is labeledwith FAM as indicated in 4B.

FIG. 4B shows MspJI-digestion on fully and hemi-methylated oligosubstrates.

-   -   Lane 1, no methylation, top strand labeled;    -   Lane 2, no methylation, bottom strand labeled;    -   Lane 3, both top and bottom methylated, top strand labeled;    -   Lane 4, both top and bottom strand methylated, bottom strand        labeled; cleaved products with sizes of 8 nt and 7 nt suggest        wobbling cuts in the bottom strand;    -   Lane 5, top strand methylated, top strand labeled;    -   Lane 6, top strand methylated, bottom strand labeled; as in Lane        4, cleaved products with sizes of 8 nt and 7 nt suggest wobbling        cuts in the bottom strand;    -   Lane 7, bottom strand methylated, top strand labeled; and    -   Lane 8, bottom strand methylated, bottom strand labeled.

As a control, markers are run on the right side of the gel.

FIG. 4C shows an oligonucleotide sequence having a CpG and enzymaticcleavage sites that would yield a fragment with the CpG in a centrallocation.

FIG. 4D shows digestion of the oligonucleotide shown in FIG. 4C usingMspJI in the presence or absence of activator DNA. Reactions were donein 10 μl at 37° C. for 1 hour. The oligonucleotide is 1 pmol and MspJIis 0.4 μg in each reaction. In reactions with activator, 1 μl of stock(15 μM) was added into the 10 μl reaction. 5 μl of reactions were takenout and stopped at different time points and resolved on 20% nativepolyacrylamide gel.

-   -   Lane 1, DNA only;    -   Lane 2, digestion reaction without activator at 30 min;    -   Lane 3, digestion reaction with activator at 30 min;    -   Lane 4, digestion reaction without activator at 1.5 hour; and    -   Lane 5, digestion reaction with activator at 1.5 hour.

FIG. 5 shows the sequence analysis of MspJI cleavage site positions ondifferent m5C methylated sites. pBR322DNA (dcm−) was methylated usingvarious methyltransferases. The methyltransferases are shown under thecolumn heading “methylase”. The run-off sequencing pattern is shownunder the column “examples of sequencing chromatogram” (SEQ ID NOS:2-6).The deduced cleavage patterns are shown in column “MspJI cleavage site”.

FIGS. 6A-6C show characteristics of the novel enzyme family.

FIG. 6A shows a double-stranded DNA with a centrally located modifiedcytosine, which is a representative fragment of the set ofoligonucleotides obtained when a large DNA is cleaved with a member ofthe novel enzyme family.

FIG. 6B shows a DNA sample of human genomic DNA on a polyacrylamide gelin which the set of oligonucleotide fragments shown in FIG. 6A appear asa coherent band. The 32 bp band represents the pool of short fragmentscontaining methylated CpG sites from the genome. This pool can bepurified and directly put into the Next-Generation sequencing platformsfor methylome analysis.

FIG. 6C shows a comparison of cleavage products generated by variousmember enzymes in the MspJI family. The first lane contains DNA markers.All subsequent lanes contain the digests of CpG-methylated Hela genomicDNA, each with a different MspJI family member enzyme. Lanes 1-3 and 5show a band that corresponds to about 32 nucleotides in length (arrow).In lane 4, RlaI recognizes CCWGG and cleaves on either side of therecognition site to provide a centrally located modified cytosine.However, this sequence is not found in Hela genomic DNA.

-   -   Lane 1 shows the cleavage product from MspJI;    -   Lane 2 shows the cleavage product from Frankia 5336;    -   Lane 3 shows the cleavage product from Lpg 1234;    -   Lane 4 shows the cleavage product from RlaI;    -   Lane 5 shows the cleavage product from AspBHI; and    -   Lane 6 shows DNA only.

FIGS. 7-1 to 7-8 shows the amino acid sequence alignment ofrepresentative members of the MspJI family of enzymes. Residuesconserved in 5 or more members of the alignment are indicated in the topline (“Conservation”). Secondary structure prediction is listed at thebottom (“Consensus_ss”). Secondary structural elements are: e, β sheet;h, α helix.

FIGS. 8-1 to 8-2 shows a bioinformatics analysis of CG-centeredsequences of different lengths in three organisms. Total number of CGsites are listed for each organism for human, mouse and Arabidopsisgenomes. Columns report the number of CG-centered sequences that aredistinct (differ in sequence), the number that are unique (occur insingle copy), the fraction of the total such CG-centered sequences thatare unique and the fraction of distinct CG-centered sequences that areunique (single copy).

DETAILED DESCRIPTION OF THE EMBODIMENTS

A novel family of modification-specific DNA cleavage enzymes has beenfound where the members of the family recognize a modified nucleotide indouble-stranded DNA and then cut at a non-random distance downstream (3′direction) from the modified nucleotide. One of the unique properties ofthese enzymes is that they are capable of releasing short DNA fragmentscontaining a modified nucleotide directly from large DNA includinggenomic DNA. These enzymes are capable of generating double-strandedbreaks in the DNA on both strands when a modified nucleotide is presentin opposing positions on each strand. When DNA contains a modifiednucleotide on one strand only, double strand breaks occur on one side ofthe modified nucleotide. The location of the modified nucleotide in alarge DNA can thus be deduced by cloning the cleavage products and/or bysequencing. Using ultra high throughput sequencing platforms, it ispossible to identify and map modified nucleotides such as methylatedcytosines or hydroxyl-methylated cytosines in a reliable and quickmanner.

“Modified” nucleotide is intended to refer to any nucleotides thatcontain an extra chemical group such as a 5-hydroxymethyl or 5-methylgroup. For example, a “modified” cytosine generally arises in mammaliangenomes as a CpG and in plant genomes as a CNG which, because of thesymmetry, make possible methylation on both strands at the sameposition. Hydroxymethylcytosine has been recognized as a constituent ofhuman DNA (Tahiliani et al., Science 324(5929): 930-5 (2009);Kriaucionis and Heintz, Science 324(5929): 929-30 (2009)).

“Large DNA” is intended to refer to any naturally occurring or syntheticDNA having a size greater than 100 nucleotides up to a size of a genome.

“Similar size” with reference to a “set” of oligonucleotide fragments isintended to refer to fragments that vary no more than about ±5nucleotides in length. However, different “sets” of fragments may have asize range of 5-50 nucleotides.

“Centrally positioned” is intended to correspond to a location of amodified nucleotide on one strand which is approximately centered in thesame strand of a double-stranded fragment. The location is generallywithin 5 nucleotides of the center determined by counting thenucleotides from either end of the fragment.

“N-terminal domain” refers to a region extending to about 50% of theamino acid sequence of the protein. In an embodiment of the invention, aconserved region within the N-terminal domain corresponds to amino acids81 to 224 of SEQ ID:NO:22 (MspJI) and a conserved region within theC-terminal domain corresponds to amino acids 300 to the C-terminus ofthe protein (SEQ ID NO: 22 (MspJI)).

A “set of oligonucleotide fragments” of similar size obtained bycleavage of a large double-stranded DNA refers to the fragmentsresulting from cleavage of the large DNA on both sides of a modifiednucleotide when the modified nucleotide is located on one strandapproximately opposite to another modified nucleotide on the secondcomplementary strand.

An “enzyme preparation” is intended to refer to a reagent and notsomething occurring in its natural state in vivo.

If a genome that consists of multiple large DNAs (e.g., chromosomes) iscleaved, each large DNA will give rise to a set of oligonucleotidefragments of similar size. A mixture of fragments obtained from cleavageof an entire human genome can be considered as a plurality of sets ofoligonucleotide fragments, each set derived from a chromosome or as asingle set of fragments depending on the context. In an embodiment, theset of oligonucleotides comprises at least 6 oligonucleotide fragmentswith different DNA sequences. For example, the set of oligonucleotidesmay comprise at least 10 oligonucleotides with different sequences or atleast 20 oligonucleotides with different sequences. In one embodiment, acloned double-stranded DNA can be enzymatically modified at a targetnucleotide at one site for example, modification of a cytosine at a CpGon both strands. In this example, double strand cleavage by a member ofthe MspJI enzyme family will occur on both sides of the modifiedcytosine at a non-random distance. The set of oligonucleotides willconsist of similarly sized fragments with a centrally located modifiednucleotide.

Members of the newly described MspJI family of enzymes have beenidentified from microbial sources although enzymes in the family are notlimited to those found in microbes. A BLAST search has shown that thenumber of sequences identified in the DNA database containing genomesfrom all living sources that encode proteins in the family as definedherein is relatively small. Sixteen homologs are shown in FIGS. 1C, 1Dand 7-1 to 7-8 and their % sequence identity and % homology (similarity)are shown in Table 2.

Enzymes capable of recognizing a modified nucleotide and cleaving theDNA at a non-random distance from the modified nucleotide have beenfound to share sequence motifs in the N-terminal domain. These enzymeshave been found to be capable of uniquely cleaving the DNA on both sidesof the modified nucleotide to produce a fragment of a non-random size. Amodification on the 5 position of cytosine (m5C) in a eukaryotic genomeis most commonly associated with regulation of gene expression.Embodiments of the invention may encompass enzymes capable ofrecognizing a modified cytosine at a CpG site, the exocyclic N4 positionof cytosine (mN4C) or a modified nucleotide other than cytosine such asadenine, for example, the exocyclic N6 position of adenine (mN6A) wheresuch enzymes cleave on either side of the modified recognition sequence.

The family of enzymes defined herein by a conserved sequence domain andcertain functional features include derivative enzymes or variants thathave sequence modifications outside or inside the recognition and/orcatalytic domains. Additionally, recombinant derivative enzymes orenzyme variants are included in the family that may be fused to a secondprotein which serves as a label, tag or marker (U.S. Pat. No. 5,643,758)or may contain a substitution which acts as a label such as occurs witha selenocysteine substitution (see U.S. Pat. No. 7,141,366). In additionto the above described family of enzymes, derivative enzymes and enzymevariants are contemplated in which the catalytic domain is modified orabsent such that the N-terminal domain acts as a methylated DNA orhydroxymethylated DNA binding domain.

The use of the MspJI family of enzymes to generate a set ofoligonucleotide fragments may rely on a single enzyme or may include aplurality of enzymes where some or all of the enzymes are members of theMspJI family or are derived from members of the MspJI family.

The members of the newly defined family, before chemical modification ormutation, can be defined structurally by one or more of the featureslisted below.

-   -   (a) non-heteromeric;    -   (b) recognition and cleavage functions in a single open reading        frame;    -   (c) the coding sequence and protein sequence do not contain        methyltransferase motifs;    -   (d) at least 90% sequence homology to a conserved motif in the        N-terminal domain which is WXD(X)₁₀YXGD; and    -   (e) common secondary structure elements that encompass the        conserved motif.

FIG. 1B and FIGS. 7-1 to 7-8 show an embodiment of the enzyme family inwhich the overall order of the secondary structure elements that buildthe catalytic core ishelix(H1)-helix(H2)-sheet(S1)-sheet(S2)-sheet(S3)-helix(H3)-sheet(S4)-helix(H4)(see, for example, FIGS. 7-1 to 7-8 where h=helix and a series of erepresents a β-pleated sheet). The conserved FE is in an α-helix H2; thefirst conserved aspartic acid (D) is in a hinge region between two βsheets S1 and S2; the second conserved aspartic acid is in β sheet S2;the conserved (Q/E)XK is in a β sheet S3.

Members of the family may be identified by a BLAST search using asequence selected from SEQ ID NO:7-22 or a related sequence. The hitsare then further searched for the above specified consensus sequence inthe N-terminal domain and also optionally searched for at least 90%sequence homology or identity in the C-terminal domain of a consensussequence FEX₂₀₋₃₀DX₂₋₄DX₁₉₋₂₂(Q/E)XK. Optionally, the conserved sequencein the N-terminal domain can be extended to greater than 90% sequencehomology to WXD(X)₆G (X)₃YXGD(X)₁₀₋₁₅GN(X)₂L X₁₀₋₂₀PX₃F and/or greaterthan 90% sequence homology with FE(X)₂A(X)₁₅₋₁₈T/SX₄DGGXDX₂G/LX₁₅₋₂₀E/QAK.

The selected sequences may then be expressed by techniques known in theart, for example in vitro transcription-translation (PURExpress™, NewEngland Biolabs, Inc. (NEB), Ipswich, Mass.) or by cloning into amicrobial host lacking modified bases such as #ER2655 (NEB Express,#C2523, NEB, Ipswich, Mass.) and assayed for cleavage of DNA containingmodified nucleotides to produce oligonucleotide fragments of a definedsize and/or containing a centrally located modified nucleotide.

Antibodies may be raised to members of the newly defined family ofenzymes (MspJI enzyme family) using standard techniques for generatingmonoclonal or polyclonal antibodies. These antibodies or fragmentsthereof may be used for in situ-labeling of a member of an MspJI enzymefamily bound to the modified large DNA. The enzyme may be mutated sothat the cleavage function is inactivated or removed. In this context,the fragments may then be separated by binding to affinity matricescapable of antibody binding.

Functionally, MspJI was identified as a DNA sequence in the databaseadjacent to a methylase gene sequence and was hence named anendonuclease gene. However, when it was expressed as a protein, it wasfound to be inactive using standard assays for determining restrictionendonuclease activity. This would have normally terminated any furtherstudy but for the fortuitous discovery described here that whenincubated with DNA from a Dcm+ strain of E. coli, the enzyme was active,while it was inactive when tested on DNA from a Dcm− strain of E. coli.When the enzyme was incubated with eukaryotic DNA that is known tocontain modified cytosines such as human genomic DNA, a smear of highmolecular weight DNA was observed on polyacrylamide gels together with aclearly visible band containing fragments that correspond to a size ofabout 32 base pairs (see FIG. 6B).

A family of related enzymes were identified (FIGS. 7-1 to 7-8) and DNAcleavage by representative examples of these enzymes were tested withhuman genomic DNA. The tested enzymes yielded a set of similarly sizedoligonucleotide fragments of about 32 nucleotides as can be observed onthe gel in FIG. 6C.

The newly defined family of enzymes described here is of particularinterest for reasons that include their ability to recognize nucleotideresidues modified at the 5 position and to produce a set ofoligonucleotide fragments where cleavage occurs at a substantially fixeddistance downstream of the enzyme recognition site on the DNA (see FIGS.6A, 6B and 6C). In embodiments of the invention, the cleavage distancefrom the modified sites conform to the following rules:

-   -   (1) For double-stranded DNA with a palindromic m5 CpG or other        modified nucleotide on both strands in close proximity, a        double-stranded break may be generated on each side of the        modified nucleotide to generate fragments of similar size. In        one embodiment, the distance between the cleavage site on one        strand with a modified CpG was found to be 12 bases and the        distance to the cleavage site on the opposite strand from the        m5C was found to be 16 bases (MspJI) including a 4 base overhang        resulting in oligonucleotide fragments of 32 bases in length.    -   (2) For hemi-modified double-stranded DNA, a double-stranded        break occurs at a position which is 3′ downstream from the        modified nucleotide. The distance from the cleavage site on the        same strand to the modified nucleotide is constant (for example        for MspJI the distance is 12 bases and the distance from the        cleavage site on the other strand to the modified nucleotide is        16 bases). Sites of hemi-modification in the DNA can be detected        by ligating an oligonucleotide containing a site recognized by        an MmeI-like enzyme (such as MmeI, see U.S. Pat. No. 7,115,407)        to a hemi-modified DNA cleaved at one side at a site 16        nucleotides from the modified nucleotide. The oligonucleotide        may include four degenerate nucleotides at the 5′ end of the        MmeI-site oligonucleotide to allow annealing to the 4 base        extension on the bottom strand. Alternatively, a blunt-ended        oligonucleotide might be used such that the single strand region        at the 4-base extension is filled in using standard molecular        biology techniques. The MmeI-like enzyme will cleave 18 or 19        nucleotides upstream which for MspJI cleavage fragments is about        2 nucleotides upstream of the modified fragment. Fragments        produced in this manner can be sequenced and the position of the        hemi-modified nucleotide in the DNA determined.

The number of CpG sites in genomes from human, mouse and Arabidopsis hasbeen determined using bioinformatics. If the genomes of these organismsare then cleaved into various length fragments from 24 bases to 60 basescontaining a centrally positioned CpG, then the fragments with uniquesequences represent between 71% and 91% of the total unique sequence inhumans according to increasing size, 83% to 90% in mice and 89% to 95%in Arabidopsis by the same criteria.

If those sequences of a defined length which have a distinct sequenceare distinguished from the total sequence and separately analyzed, then96%-98% of fragments of size 24-60 nucleotides will match a single locusin the human genome. In the bioinformatic analysis provided in FIGS. 8-1to 8-2, if the fragment length is 60 nucleotides, then there are26,185,493 fragments that contain a centrally positioned CpG of which25,538,480 have distinct sequences and 98% of these match a single locusin the genome (see FIGS. 8-1 to 8-2).

Hence, there is significant informational value in a set ofoligonucleotide fragments generated by an enzyme in the newly definedfamily where the enzyme recognizes a modified nucleotide and cleaves theDNA at a distance from the modified nucleotide to preferably generatefragments of a similar size. The data shows that a large fraction ofsuch fragments are highly likely to map to a single locus in the genome.This makes possible for the first time a simple and efficient method forcreating a methylome. Consequently, high throughput sequence analysiscan yield the location of the majority if not all of the actual modifiednucleotides in the genome rapidly and easily.

In an embodiment of the invention, screening assays are described fordetermining modification-specific cleavage activity of an enzyme (seeExample 1). These assays are not intended to be limiting. In oneembodiment, selected host cells were transformed with a plasmidcontaining a specific DNA methyltransferase gene. The expressedmethyltransferase then methylated the host genome at specific sites.Hundreds of methyltransferases with varying defined sequencespecificities have been described in the literature (see for exampleREBASE®, a publicly available online database maintained by New EnglandBiolabs, Ipswich, Mass.). Any of these methyltransferases with differentmethylation specificities can be used for screening purpose.Introduction of a compatible plasmid expressing a gene withmodification-dependent cleavage activity able to act on the host'smodification pattern would reduce or eliminate the viability of thesetransformed cells, leading to a low transformation plating efficiency.Non-methylated hosts would show high plating efficiency in a paralleltransformation with the methylation-specific endonuclease gene. Thus,this test would confirm the modification-dependent cleavage property ofthe encoded gene product.

It was found that the activity of the enzymes in the MspJI family can beenhanced in the presence of a double strand DNA activator preferablyhaving a length of less than 16 bp and containing a modified dcm site(for example, dcm(C^(5m)CWGG) site). A 30 bp cleavage-resistant DNAactivator containing phosphorothioate linkages at the cleavage site alsostimulated the enzyme reaction.

Determining the level of methylation or hydroxymethylation of DNAsamples is important for epigenetic studies. Epigenetic regulation ofthe genome includes chromatin remodeling which may be accomplished bythe addition of methyl groups to the DNA, mostly at CpG sites to convertcytosine to 5-methylcytosine, and its reversal possibly viahydroxymethylcytosine. Methylation of cytosines in the eukaryotic genomemay persist from the germline of one parent into the zygote marking thechromosome as being inherited from this parent (genetic imprinting).Moreover, large changes in methylation occur following zygosis and intissues of the developing organism (Morgan et al. Hum Mol Genet 14 SpecNo. 1:R47-58 (2005)). In addition, methylation in some regions of thegenome may vary in response to environmental factors (Li, et al Cell 69(6): 915-926 (1992)). Differences in methylation pattern may be criticalindicators of inappropriate developmental processes for example forembryonic stem cells (Brunner et al. Genome Research 19:1044-1056(2009)).

Certain enzymes (such as DNMT1) have a high affinity for the m5C. Ifthis enzyme reaches a “hemi-methylated” portion of DNA (wheremethylcytosine is in only one of the two DNA strands), the enzyme willmethylate the other half.

DNA methylation occurs in repeated sequences, and helps to suppress theexpression and mobility of transposable elements (Slotkin, et al. NatRev Genet. 8(4):272-85 (2007)). Because of spontaneous deamination,5-methylcytosine can be converted to thymidine; hence, CpG sites arefrequently mutated and thus become rare in the genome, except at CpGislands where they remain unmethylated. Deamination in this situationconverts cytosine to uracil. Diagnostic changes in methylation patternhave the potential to detect increased frequencies of permanent geneticmutation. Methylation in the human genome has been studied in cancercells for purposes of exploring therapies (see for example, Gargiulo, etal. The International Journal of Biochemistry & Cell Biology 41:127-35(2009); and Gronbaek, et al. Basic Clin Pharmacol Toxicol 103:389-96(2008)).

Embodiments of the invention significantly advance the ability to mapmodified nucleotides in a genome to generate a map (methylome). A humanmethylome would facilitate studies on interpersonal phenotypicvariations in whole organisms and in individual cells and can yielduseful information on development, aging and disease. From thisinformation, it may be possible to determine susceptibility for diseasessuch as cancer even before a pathology appears and to design appropriatetreatments with the possibility of providing powerful diagnostic testsand therapeutic agents.

The identification of a family of enzymes with novel properties and thecreation of novel oligonucleotide fragments permits a description of thestatus of the human methylome by allowing the isolation of a set or setsof oligonucleotide fragments that provide a concentration of modifiedbases found in the human genome. Isolation of the set(s) of fragmentscan be facilitated by gel electrophoresis, solid phase affinity-bindingor other means. Methylome analysis can be aided by the addition of acontrol that may include treating the genome with M.SssI, whichmethylates substantially all CpG dinucleotides in the genome.(Yegnasubramanian et al. Nucleic Acids Res 34:e19 (2006)).

The set(s) of oligonucleotide fragments resulting from enzyme cleavagecan be sequenced using high throughput sequencing methods of the sortthat are currently available using NextGen sequencing methods toidentify and map modified cytosine nucleotides in a DNA. This approachgreatly simplifies the generation of a methylome for any large DNA orgenome such as a mammalian genome. Selection of specific oligonucleotidecleavage products for rapid diagnostic methods to particular regions ofthe genome can determine the abnormal presence or absence of modifiedcytosines correlated with a disease such as cancer. Specificoligonucleotides may be used to determine a particular phenotype for anindividual. For example, hybridization of a set of fragments to adefined sequence or set of sequences presented on a solid surface (arrayhybridization) or tagged in a solution (or visa versa) can revealdiscrepancies with a standard set of fragments that characterize themethylome. qPCR or array hybridization may also be used to interrogateone or more known locations of interest for abundance. The modifiednucleotide or binding molecule may be labelled with a fluorescent orchemiluminescent tag or other labelling methods known in the art tofacilitate detection.

Modified nucleotides in the genome may be identified in situ using amutant enzyme member of the newly defined family having an inactivatedcleavage site. By visualizing the binding sites of the mutant enzyme,the location of the modified nucleotides can be determined.

The members of the newly defined family of enzymes may be geneticallyengineered so as to form recombinant proteins for large scaleproduction. The purification of recombinant proteins may be facilitatedby the formation of fusion proteins such that the enzyme is fused to anaffinity tag which has an additional use. For example, if the tag isbiotin, a His peptide, chitin-binding domain or maltose-binding proteinor another substrate-binding domain, the member of the family may beisolated on an affinity matrix either directly itself acting as amethyl-binding domain or by binding to an antibody affinity matrix or bymeans of the affinity tag. The recombinant protein either alone,modified or fused to a tag may be fluorescently labeled for imagingpurposes.

Where the enzyme kinetics are single turnover, low turnover or theenzyme lacks catalytic activity altogether, the enzyme or enzyme fusionprotein while bound to a modified nucleotide-containing fragment mayalso be bound directly to an affinity matrix to separate theoligonucleotide fragments containing modified nucleotides from theremaining fragments for sequencing or for diagnostic tests.

The experimental protocols provided below in large part for MspJI arenot intended to be limiting. One of ordinary skill in the art couldemploy the experimental design as provided below to any additionalmember of the newly defined family.

EXAMPLES Example 1 Methylation-specific DNA Cleavage Activity of MspJIEnzyme Family

Production of the Enzymes

Recombinant members of the MspJI enzyme family were expressed in dcm−strain ER2566 and purified until substantially homogeneous usingmultiple chromatography steps. The enzymes which had an N-terminal 8×HisTag were first purified on a HiTrap Heparin HP column (GE, Piscataway,N.J.), then a HisTrap HP column (GE, Piscataway, N.J.), and finally aHiTrap SP column (GE, Piscataway, N.J.). The purification procedurefollowed the manufacturer's recommendation. The cleavage activity of theenzyme fractions were assayed on lambda DNA (which is partiallydcm-methylated). To further improve expression levels, the DNA encodingthe enzymes can be codon-optimized.

Determining Cleavage Patterns of MspJI Enzyme Family by a ScreeningAssay

The assay may include one or more of the following steps:

-   -   1. Methylate a target nucleotide in a synthetic or naturally        occurring large DNA optionally having a known sequence. For        example, lambda DNA can be used which is partially        dcm-methylated at CmCWGG sites and XP-12 phage genomic DNA whose        cytosines are completely replaced by 5mC cytosines.    -   2. React the large DNA with an MspJI enzyme family.    -   3. Size-separate the cleavage products for example using a        polyacrylamide gel.    -   4. Sequence a set of oligonucleotide fragments of similar size        to determine the position of the modified nucleotide; and    -   5. Optionally map the fragment sequence on the large DNA        sequence.    -   An elaboration of step (1) includes using different large DNA        preparations which have been reacted with different        methyltransferases for modifying the DNA in vitro. These        substrates are used to identify substrate specificities.

The products are analyzed by 1% agarose gel electrophoresis and can bevisualized by ethidium bromide. For example, M.HpaII (NEB, Ipswich,Mass.) can produce CmCGG-modified DNA. Plasmid DNA-digestion can bemonitored by 1% agarose gel electrophoresis and visualization usingethidium bromide staining. Alternatively, synthetic double-strandedoligonucleotide containing a modified site can be used in which anymethylated sites can be easily created, independent of the availabilityof the methyltransferases. Modified nucleotides in palindromic sites ofinterest include for example, NmCGN, mCNG, NGmCN, GNmC etc., where N isA, T, G or C. In addition to fully-methylated oligonucleotides,oligonucleotides with hemi-methylated sites can be tested in this way.Other types of modification, such as 5-hydroxymethylated cytosine and5-glucosylated-hydroxymethylated cytosine, can be either directlyincorporated into the oligonucleotides during synthesis or by furthermodification of hydroxymethylated cytosine residues with bacteriophageT4 glucosyltransferase.

To determine cleavage sites, substrate oligonucleotides are labeledeither at their 5′ end or 3′ end with ³³P. Cleavage products are run ona 7M-urea 20% polyacrylamide denaturing gel to single nucleotideresolution and analyzed.

Characterizing MspJI Enzyme Family Members by an In Vivo Screening Assay

The ER1992 strain with endogenous methylase gene dcm, which methylatesthe inner cytosine in CCWGG sites to CmCWGG and serves as a targetsubstrate for an enzyme with the desired cleavage activity, and ER2566,with a dcm− genotype with no 5-methyl cytosine and not subject tocleavage by a methylation-specific enzyme, were used to screen formethylation-specific activity of a novel recombinant restrictionendonuclease.

Measuring the Activity of MspJI Enzyme Family Members by an In VitroAssay

A plasmid is used which contains only two methyl-C's separated by 1 kbof intervening sequence. This cleaves leaving three fragments, theplasmid backbone 3 kb, the insert 1 kb and the two 32 bp fragments. Whenthis digest goes to completion, the uncut plasmid disappears andsubsequent appearance of the 1 kb and backbone bands is easilyquantifiable on an agarose gel. This plasmid is transformed into adam-dcm− strain and purified as an assay substrate. Such plasmids aredescribed in Stewart, F., et al. Biological Chemistry 379:611-616(1998).

Determining Specificity of MspJI Enzyme Family Members for ModifiedBinding Sequences Versus Unmodified Binding Sequences Using an In VitroAssay

The in vitro activity of MspJI was assessed on a variety of methylatedand non-methylated DNA substrates, as shown in FIG. 2A for thedcm-methylated plasmid DNA pBR322. MspJI showed endonuclease activity(FIG. 2A, Lanes 1 and 2) where this endonuclease activity was DNAmethylation-dependent. In contrast, MspJI did not act on pBR322 withoutdcm-modification (FIG. 2A, Lanes 5 and 6). By using the restrictionenzyme BstNI (CC⇓WGG), which is insensitive to m5C methylation, in adouble-digestion assay, cleavage sites on pBR322(dcm+) by MspJI wereshown to be at or close to the dcm sites (FIG. 2A, Lanes 2, 3 and 4).The double digest did not alter the BstNI pattern, suggesting that MspJIdid not cleave at non-BstNI sites.

In addition to the m5C-modified DNA tested above, MspJI did not exhibitendonuclease activity on M.TaqI- (TCGmA) or dam- (GmATC) methylatedpBR322(dcm−) DNA. This confirmed that MspJI did not target m⁶-adeninemethylated DNA, consistent with the fact that the MspJI gene can bemaintained and expressed in a dam+ strain (ER2566, NEB, Ipswich, Mass.).Moreover, MspJI does not apparently act on N⁴-methylcytosine-containingplasmid DNA, as can be determined by using M.BstNI (CCWGG, a N4-cytosinemethylase) methylated DNA.

Assaying Activity of MspJI Enzyme Family Members on DNA Substrates whichContain 5-hydroxymethylcytosine or 5-glucosyl-hydroxymethylcytosine

Wild-type T4 phage DNA with glucosylated cytosines and the DNA from a T4α gt57 β gt14 (a mutant which has defective glucosyltransferases andtherefore contains hydroxymethylated cytosines in DNA hereafter T4gt)were used as substrates (FIG. 2C, Lanes 1 and 2). MspJI was able todegrade T4 gt DNA (FIG. 2C, Lanes 8-11) and was inactive on glucosylatedDNA (FIG. 2C, Lane 7). For comparison, activity of anothermodification-dependent endonuclease, McrBC (FIG. 2C, Lanes 3 and 4) anda typical type IIP restriction enzyme MspI (FIG. 2C, Lanes 5 and 6) withthese modified DNA substrates were shown. McrBC, which recognizes pairsof (A/G)^(m)C separated by 40˜3000 base pairs, also exhibited nucleaseactivity on the hydroxymethylcytosine-containing DNA but not on T4wild-type DNA, while MspI was inactive with respect to both substrates.Note that MspJI was able to degrade T4 gt DNA to a greater extent thanMcrBC, which can be explained by its broader recognition sequence thanMcrBC. Overall, it appears that MspJI specifically targetscytosine-modified DNA with 5-CH₃ or 5-CH₂OH addition on the pyrimidinering.

Determining Substrate Sequences Around a Cleavage Site

MspJI-digested DNA samples with different methylated sites weresubjected to capillary sequencing and the cleavage sites were deducedfrom the location at which peaks are reduced in height near themethylated sites in the sequencing chromatograms (examples shown in FIG.5). Positions of cleavage occur at locations where the sequence signals(peaks) are reduced in height. In many cases, a non-templated adenine isadded as the polymerase runs off the DNA, and location of such a “runoffpeak” adenine is additional evidence for the location of cleavage. Oneobservation on the sequencing chromatogram data was that cleavage sitesoccur at a site distant from the methylated sites. FIG. 5 also displaysthe deduced cleavage pattern of MspJI on different methylated sites.Another observation was that the height reduction in sequencing peaksand addition of adenine not present in the substrate were generallypresent on both sides of the methylated sites. The response in thechromatogram on both sides demonstrated that MspJI cleaved the DNA eachside of the methylated binding sequence. This is consistent with thesymmetry of the methylated binding sites. The presence of two run-offpeaks is evidence for two independent cleavage events on the samestrand.

It was concluded that MspJI recognized the m5C on one strand and thencut 12 nucleotides 3′ downstream on the same strand and 16 nucleotidesdownstream on the complementary strand to leave a 4-base 5′ overhang.Similarly, when the m5C on the complementary strand was recognized, thesame pattern of cleavage was observed demonstrating that twodouble-stranded breaks around the same recognition site released thefragment with the methylated site in the middle. The exact length ofthat fragment depended on the distance between the methyl groups on thetwo strands. In the case of HpaII methylated sites (Cm5CGG) or HhaImethylated sites (Gm5CGC), the length of the fragment excised from theDNA substrate was expected to be 32 nucleotides including the two 4-base5′ overhangs.

Comparing the Activity of an MspJI Enzyme Family Members onFully-methylated and Hemi-methylated DNA

To investigate whether MspJI is active on hemi-methylated DNAsubstrates, which can arise during replication, FAM-labeled syntheticsubstrates were used in a digestion assay (FIG. 4A). FIG. 4A indicatesthe expected cleavage sites and product sizes and FIG. 4B shows thedigestion reactions resolved on a 7M urea 20% polyacrylamide denaturinggel. The m5C for interrogation is at an M.HpaII site (CmCGG) in theoligos. Null-methylation, full-methylation, hemi-methylation on the topstrand or bottom strand were tested and the cleavage events on the topstrand and bottom strands were observed by labeling them individually,as shown in FIG. 4B.

On fully-methylated DNA, MspJI makes cuts on both sides of themethylated site. On the top strand, it cleaves on either side of themethylated site, resulting in 40 bp (from cut Lt) and 11 or 12 bp (fromcut Rt) fragments (FIG. 4B, Lane 3). Symmetrically, on the bottomstrand, it cleaves twice and generates a long fragment of 36 nt (fromcut Lb) and a shorter fragment of 7 or 8 nt (from cut Rb) (FIG. 4B, Lane4).

On the hemi-methylated substrates, strand methylation status dictatesthe side of the cleavage, so that double-stranded breaks only occur onthe 3′ side of the strand containing the methylated base. For example,for substrate with only top strand methylation, each cleavage event isat the 3′ side of the 5mC so that only shorter fragments are observed(FIG. 4B, Lanes 5 and 6). The same applies to the substrate with bottomstrand methylation where only longer fragments are seen (FIG. 4B, Lanes7 and 8). The results show that each m5C is associated with two cuts onthe same side and such association is symmetrical. Thus, while notwishing to be bound by theory, it is proposed that MspJI recognizes eachhalf of the methylated site separately in a fully-methylated site,either the top or the bottom strand, and that the half site thendictates the directionality of the cleavage.

Characterizing the MspJI Enzyme Family Members with Shared Conserved DNASequences, Secondary Sequence Motifs, and Binding and CleavageProperties

By using the amino acid sequence of a member of the MspJI family such asMspJI as the query sequence, a PSI-BLAST search (Altschul et al.,Nucleic Acids Res 25:3389-3402 (1997)) against GenBank retrieved morethan 100 hits with significant sequence homology. Sixteen genes amongthe top hits had significant similarity to MspJI throughout the sequencelength. In FIG. 1D, a partial multiple sequence alignment is providedaround the conserved catalytic motif inside the MspJI subfamily. Thesignificance of the conserved catalytic motif is shown by thesite-directed mutagenesis experiments, in which both D334A and Q355Amutations completely abolish the catalytic activity of MspJI.

The predicted secondary structure elements of the MspJI family weredetermined using multiple sequence alignment created by PROMALSwebserver (Pei et al. Nucleic Acids Res 35:W649-652 (2007)) (FIG. 1Bshows a schematic and FIGS. 7-1 to 7-8 the full alignment). Thestructure core of the catalytic C-terminal domain has three consecutivestrands (β1β2β3 in FIG. 1B), with the motif (Q/E)xK at the end of β3 andthe conserved residue D at the beginning of β2 (FIGS. 7-1 to 7-8) (Wahet al. Proc Natl Acad Sci USA 95:10564-10569 (1998)). The two helicesand strands in the order of α4-β4-α5-β5 after β1β2β3 forms aninteraction interface between monomers (FIG. 1B).

Determining the Role of an Activator in Improving Cleaving Activity ofMembers of the Newly Defined Family of Enzymes

An activator dimer containing double-stranded 5-methyl cytosine (e.g.,11mer, 15-mer, 19mer and 23mer) are tested to determine whetherdigestion by members of the MspJI enzyme family can be enhanced. Thesedimers are constructed by annealing two single strand oligonucleotidesor by hairpin formation of a single oligonucleotide.

The assay for the activator includes constructing self-complementaryoligonucleotides containing 5-methyl-C's at the center of variouslengths. Oligonucleotides are biotinylated at the 5′ end for subsequentremoval from reactions and 3′ amino-modified such that they cannot beligated or extended. The activators are then assayed for their abilitiesto enhance cleavage before and after streptavidin bead removal forinterference in sequencing.

Example 2 Demonstration of the Application of Enzymes in Mapping theMethylome

To analyze the methylome analysis of a mouse or a human genome, 1-2 μgsof human or mouse genomic DNA is used for the methylome analysis atsingle nucleotide resolution. The genome is digested with a member ofthe MspJI family optionally in the presence of biotin-containingactivator molecules, followed by removal of activator molecules usingstreptavidin magnetic affinity beads. The digested DNA is end-repairedusing the NEBNext™ (NEB, Ipswich) end-repair module,ethanol-precipitated and dissolved in a suitable volume of water. Thedigested genomic DNA is ligated to bar-coded SOLiD primer and P1 primerusing NEBNext™ quick ligation module (NEB, Ipswich, Mass.). The ligatedproduct is separated on a 10% TBE polyacrylamide gel and the ligatedproduct of ˜110 bps (between 100-130 bp) is excised after visualizationby ethidium bromide staining. A crush and soak or a suitable elutionmethod is used to isolate DNA for SOLiD sequencing (Applied Biosystems,Inc., Life Technologies, Inc., Carlsbad, Calif.). MspJI for example doesnot distinguish between methylated and hydroxymethylated cytosineresidues for cleavage, thus the sequencing data will result in analysisof the whole methylome.

Determination of the Biological Role of 5-Hydroxy Methylcytosine inMammals

The dynamic changes of DNA methylation during mouse embryonic stem cell(ES) differentiation can be identified using the newly defined family ofenzymes. Previous reports suggest that as much as 10% of the modifiedcytosines are in the form of 5-hydroxymethylcytosine. These will havebeen missed using current methodologies involving bisulfite. Thismodified adduct is complementary to guanine and is read as cytosine inpolymerase-based amplification.

Exploration of Methylomes of Other Model Organisms.

The MspJI enzyme family not only acts on mCpG, but is capable ofrecognizing and cleaving other types of methylated sites. For example,mCNG, which is present in the genomic DNA of Arabidopsis, is a naturalsubstrate for MspJI. This provides a simple method of assaying for thepresence of modified bases in any organism. For instance, digestion oftotal genomic DNA with MspJI gives a 32 bp fragment that is easy toisolate from a polyacrylamide gel. It can then be digested tomononucleotides using a standard cocktail of enzymes and the totaldigest examined by HPLC and/or mass spectrometry to identify themodified bases. A variety of organisms such as Arabidopsis, Xenopus,zebrafish, chicken, Neurospora crassa as well as genomes known tocontain unusual modifications like Base 3 found in kinetoplastidprotozoans such as Trypanosomes (Cross et al. EMBO J. 18:6573-6581(1999)) is studied by the methods described herein. Once an epigenome isconfirmed, the digested bands can be sent for high throughput sequencingusing the established protocols for human.

TABLE 1 Genomic context analysis of the MspJI subfamily Close toActivity on # Gene ID Genbank ID methylase? Species Additional notes 5mCDNA* 1 MspJI YP_001069123 Y Mycobacterium sp. JLS close to M and V genesY 2 Sbal195_0369 YP_001552810 Y Shewanella baltica OS195 2 ORFs from Mgene N/T 3 PE36_01892 ZP_01896882 Y Moritella sp. PE36 SAM-dependent N/Tmethylase 4 Spea_3849 YP_001503694 N Shewanella pealeana ATCC N 700345 5Xcc3577 NP_638923 N Xanthomonas campestris Among transposase N ATCC33913 islands 6 Lhv_0031 YP_001576608 N Lactobacillus helveticus DPCAmong transposase N 4571 islands 7 Lpg1234 YP_095265 Y Legionellapneumophila strain Close to R and M Y Philadelphia 1 genes; M gene isactive. 8 Franean1_5336 YP_001509600 N Frankia sp. EAN1pec Standalone Y9 CATMIT_00196 ZP_03681584 N/A Catenibacterium mitsuokai Unfinishedgenome N/T DSM 15897 sequence 10 Rmet_0004 YP_582159 N Ralstoniametallidurans CH34 Standalone N 11 Bcenmc03_0011 YP_001763314 YBurkholderia cecepacia MC0-3 Close to M and V N/T genes 12 AspBH1YP_931859 Y Azoarcus sp. BH72 Close to M and V Y genes 13 RlaIZP_03168528 N Ruminococcus lactaris ATCC Close to V gene Y 29176 14Xccb100_0619 YP_001902024 N Xanthomonas campestris strain Amongtransposase N/T B100 islands 15 ZP_03855940 ZP_03855940 Y Veillonellaparvula DSM 2008 Close to M gene N

TABLE 2 Similarity and identity of sequences in the familyidentity/similarity AspBHI Bcenmc03_0011 CATMIT_00196 Franean1_5336AspBHI 100/100 36.5/47.4 41.3/57.7 40.1/52.4 Bcenmc03_0011 36.5/47.4100/100 30.8/43.6 27.9/40.6 CATMIT_00196 41.3/57.7 30.8/43.6 100/10035.9/51.1 Franean1_5336 40.1/52.4 27.9/40.6 35.9/51.1 100/100 MspJI25.6/38.0 18.9/28.7 20.2/37.1 22.3/34.5 PE36_01892 22.8/38.3 16.5/29.124.7/41.2 23.2/37.0 RlaI 42.3/58.2 32.8/45.9 40.9/61.8 36.6/51.7Rmet_0004 38.6/52.7 32.3/48.5 33.1/47.3 35.5/46.5 Sbal195_0369 22.6/36.716.4/28.8 24.7/42.0 22.8/35.0 SgriT_16873 45.7/60.9 30.2/42.8 37.0/55.452.1/64.2 Spea_3849 21.5/35.2 17.5/29.7 24.8/43.5 23.6/36.2 Xcc357724.2/37.9 19.6/30.2 25.7/38.7 22.4/32.0 gi|227372459| 39.1/58.029.6/44.8 42.3/60.6 31.9/47.7 ref|ZP_03855940.1| V. par gi|26010182922.2/36.1 19.4/34.6 22.3/39.1 21.4/30.3 DSM 20075 lhv_0031 21.3/34.817.3/30.8 23.2/40.7 21.7/31.0 lpg1234 42.6/62.9 28.4/43.8 37.8/57.037.0/52.4 identity/similarity MspJI PE36_01892 RlaI Rmet_0004 AspBHI25.6/38.0 22.8/38.3 42.2/58.0 38.6/52.7 Bcenmc03_0011 18.9/28.716.7/29.8 32.8/45.9 33.4/47.4 CATMIT_00196 20.2/37.1 24.7/41.2 40.9/61.833.1/47.3 Franean1_5336 22.3/34.5 23.2/37.0 36.6/51.7 35.9/46.7 MspJI100/100 36.5/53.4 23.1/40.2 22.6/35.3 PE36_01892 36.5/53.4 100/10021.6/36.8 24.1/36.7 RlaI 23.1/40.2 21.6/36.8 100/100 38.0/53.4 Rmet_000422.5/36.6 24.0/37.7 38.0/53.4 100/100 Sbal195_0369 36.3/54.4 73.8/83.324.3/41.3 24.1/35.1 SgriT_16873 25.4/39.8 26.4/40.6 41.3/57.7 37.4/50.8Spea_3849 34.3/53.9 70.6/81.7 23.9/42.0 24.2/35.2 Xcc3577 38.2/51.540.9/60.0 21.5/39.5 22.6/36.7 gi|227372459| 22.2/41.2 24.8/39.742.3/59.9 34.8/49.2 ref|ZP_03855940.1| V. par gi|260101829 29.2/43.634.6/50.9 24.1/38.0 23.8/39.2 DSM 20075 lhv_0031 30.4/46.0 36.3/53.524.8/40.0 21.5/34.9 lpg1234 21.3/37.8 26.1/40.9 41.6/58.7 49.3/62.1identity/similarity Sbal195_0369 SgriT_16873 Spea_3849 Xcc3577 AspBHI22.6/36.7 45.7/60.9 21.5/35.2 24.2/37.9 Bcenmc03_0011 16.4/28.830.2/42.8 17.5/29.7 19.6/30.2 CATMIT_00196 24.7/42.0 37.0/55.4 24.8/43.525.7/38.7 Franean1_5336 22.8/35.0 52.1/64.2 24.0/36.3 22.4/32.0 MspJI36.3/54.4 25.4/39.8 34.3/53.9 38.2/51.5 PE36_01892 73.8/83.3 26.2/40.470.6/81.7 40.9/60.0 RlaI 24.3/41.3 41.3/57.7 23.9/42.0 22.2/40.2Rmet_0004 24.1/35.1 37.1/50.4 24.2/35.2 22.6/36.7 Sbal195_0369 100/10024.0/36.5 86.5/93.1 40.6/60.7 SgriT_16873 24.0/36.5 100/100 25.4/38.223.2/32.7 Spea_3849 86.5/93.1 25.4/38.2 100/100 39.9/58.9 Xcc357740.6/60.7 23.2/32.7 39.9/58.9 100/100 gi|227372459|ref| 22.9/40.738.8/56.1 24.7/42.7 21.2/38.2 ZP_03855940.1| V. par gi|26010182936.0/50.1 23.9/36.6 35.1/49.8 31.5/48.5 DSM 20075 lhv_0031 38.6/54.925.5/38.5 36.5/53.0 33.1/51.6 lpg1234 25.1/41.3 44.3/60.1 23.5/38.019.8/40.4 gi|227372459| ref| ZP_03855940.1| gi|260101829identity/similarity V. par DSM 20075 lhv_0031 lpg1234 AspBHI 39.1/58.022.2/36.1 21.3/34.8 42.6/62.9 Bcenmc03_0011 29.6/44.8 19.4/34.617.3/30.8 28.4/43.8 CATMIT_00196 42.3/60.6 22.3/39.1 23.2/40.7 37.8/57.0Franean1_5336 31.9/47.7 21.4/30.3 21.7/31.0 37.0/52.4 MspJI 22.2/41.229.2/43.6 30.4/46.0 21.3/37.8 PE36_01892 24.8/39.7 34.6/50.9 36.3/53.526.1/40.9 RlaI 42.5/60.1 24.1/38.0 24.8/40.0 41.6/58.7 Rmet_000434.8/49.2 23.8/39.2 21.5/34.9 49.3/62.1 Sbal195_0369 22.9/40.7 36.0/50.138.6/54.9 25.1/41.3 SgriT_16873 38.8/56.1 23.7/36.4 25.3/38.4 44.3/60.1Spea_3849 24.7/42.7 35.1/49.8 36.5/53.0 23.5/38.0 Xcc3577 21.2/38.231.5/48.5 33.1/51.6 19.8/39.0 gi|227372459|ref| 100/100 100/10023.9/40.7 42.0/60.9 ZP_03855940.1| V. par gi|260101829 100/100 100/10088.2/88.2 21.3/35.6 DSM 20075 lhv_0031 23.9/40.7 88.2/88.2 100/10023.2/38.7 lpg1234 42.0/60.9 21.3/35.6 23.2/38.7 100/100

What is claimed is:
 1. A method for analyzing methylation of a genome,comprising: (a) enzymatically cleaving a sample comprising DNA from thegenome to obtain a set of similar sized double stranded fragments thatare characterized by i. staggered ends; and ii. a centrally locatedmethylated or hydroxymethylated cytosine proximal to guanine; (b)sequencing the fragments; and (c) mapping the sequences obtained in step(b) onto a sequence map of the genome, thereby determining the locationof a methylated or hydroxymethylated cytosine in the genome.
 2. Themethod of claim 1, wherein the method comprises separating the set ofsimilarly sized fragments from uncleaved DNA prior to sequencing.
 3. Themethod of claim 1, wherein the genome is a mammalian genome.
 4. Themethod of claim 1, wherein the genome is a human genome.
 5. The methodof claim 1, wherein the similar sized fragments are less than 60nucleotides in size.
 6. The method of claim 1, wherein the similar sizedfragments have a size in the range of 28-36 nucleotides.
 7. The methodof claim 1, wherein the methylated cytosine is located within 30nucleotides from one end of a fragment.
 8. The method of claim 1,wherein the centrally positioned methylated cytosine is in a CpGdinucleotide.
 9. The method of claim 1, wherein the centrally positionedmethylated cytosine is in a CNG trinucleotide.
 10. The method accordingto claim 1, further comprising: counting the distribution of CG, CHG andCHH sites (H=C/A/T).
 11. The method according to claim 10, furthercomprising annotating a whole genome map with the distribution of CG,CHG and CHH sites (H=C/A/T); sequencing depth at each methylatedcytosine; and optionally with chromosome number; chromosomal locationnumber; strand information; and enzyme recognition sites.
 12. The methodof claim 1, wherein the enzymatically cleaving is done by a restrictionendonuclease having an amino acid sequence that is at least 90%identical to SEQ ID NO:38; SEQ ID NO:29; SEQ ID NO:25; SEQ ID NO:27; SEQID NO:31; or SEQ ID NO:26.